Sphingosine-1-phosphate (S1P), a blood-borne sphingolipid, regulates many cellular functions (e.g. migration, survival) through the interaction with its G protein coupled receptors S1P1-5R. At the cellular level, S1P1R and S1P2R mediate opposite effects due to the coupling of S1P1 to the Gi-phosphatidylinositol-3- kinase (PI3K) pathway and the coupling of S1P2R to the G12/13-Rho pathway, which activates the phosphatase PTEN. While S1P1R regulates vascular permeability, angiogenesis and the inflammatory response of the endothelium, the role of S1P2R in the regulation of endothelial responses to injury remains uncertain. The main hypothesis of this proposal is that S1P2R signaling in endothelial cells is a critical modulator of vascular permeability, angiogenesis and the inflammatory responses of the endothelium by counterbalancing S1P1R- Gi-PI3K signaling (via PTEN activation) and activating the pro-inflammatory ASK-1-SAPK pathway. To test this hypothesis we propose three specific aims: Specific Aim 1: To study the role of S1P2R-Rho-PTEN pathway in the regulation of endothelial cell barrier function and vascular permeability. Molecular and cellular biology approaches will be used to characterize the mechanisms of PTEN activation by S1P2R and the role of the S1P2R-Rho-PTEN pathway in the regulation of endothelial cell permeability using human lung microvascular endothelial cells (HLMVEC), and human aortic endothelial cells (HAEC). In addition, genetic and pharmacological approaches will be used to test the role of S1P2R-Rho-PTEN pathway in the regulation of vascular permeability in vivo. Specific aim 2: To study the role of S1P2R-Rho-PTEN pathway in angiogenesis. The regulation of FOXO-1 phosphorylation, subcellular localization, ubiquitination, stability and transcriptional activity by S1P will be determined. In addition, role of the S1P2R-Rho-PTEN pathway in angiogenesis will be determined i) in vitro using gain-of-function and loss-of-function approaches, and, ii) in vivo in the Matrigel model and in a model of tumor angiogenesis, involving subcutaneous injection of the murine renal cell carcinoma line, RENCA, into syngeneic wild type, S1p2r , S1p2r-/- and Pten mice. Specific Aim 3: To determine the role of S1P2R in the regulation of the inflammatory responses of the endothelium. The mechanisms of activation ASK-1 by S1P2R will be characterized by molecular and cell biology approaches. Secondly, the role of S1P2R-G12/13-Rho-PTEN and S1P2R-G12/13-ASK-1 pathways in the regulation of the inflammatory response of the endothelium, monocyte adhesion and transendothelial migration will be studied in vitro, by gain-of-function and loss-of-function approaches. Finally, genetic approaches will be used to study the role of S1P2R and PTEN in the regulation of endothelial inflammation in a mouse peritonitis model. These studies are anticipated to provide mechanistic insights into the role of S1P2R signaling in the regulation of endothelial responses to injury. PUBLIC HEALTH RELEVANCE: S1P is a lipid, very abundant in plasma, which regulates many cellular functions through the interaction with its cell surface receptors S1P1-5R. S1P2R is present in the endothelium, which overlays the blood vessels throughout the body, and it can be activated by S1P and by pharmacological modulators. This project aims to understand how S1P interaction with S1P2R regulates the responses of the endothelium to injury. Answering these questions will help us to design new drugs that inhibit or activate S1P2R, which could be used as new therapies to alleviate the symptoms of atherosclerosis, diabetes or sepsis.